Embryonic stem cells (ESCs) represent a promising source of cells for cell replacement therapy in the context of muscle diseases; however, before ESC-based cell therapy can be translated to the clinic, we must learn to modulate cell-fate decisions in order to maximize the yield of myocytes from this systems. In order to gain a better understanding of the myogenic cell fate, we sought to define the molecular mechanisms underlying the specification and differentiation of ESCs into cardiac and skeletal muscle. More specifically, the central hypothesis of the thesis is that myogenic signalling cascades modulate cell fate via regulation of transcription factors. Retinoic acid (RA) is known to promote skeletal myogenesis, however the molecular basis for this remains unknown. We showed that RA expands the premyogenic progenitor population in mouse stem cells by directly activating pro-myogenic transcription factors such as Pax3 and Meox1. RA also acts indirectly by activating the pro-myogenic Wnt signalling cascade while simultaneously inhibiting the anti-myogenic influence of BMP4. This ultimately resulted in a significant enhancement of skeletal myogenesis. Furthermore, we showed that this effect was conserved in human embryonic stem cells, with implications for directed differentiation and cell therapy. The regulation of cardiomyogenesis by the Wnt pathway was also investigated. We identified a novel interaction between the cardiomyogenic transcription factor Nkx2.5 and the myosin phosphatase (MP) enzyme complex. Interaction with MP resulted in exclusion of Nkx2.5 from the nucleus and inhibition of its transcriptional activity. Finally, we showed that this interaction was modulated by phosphorylation of the Mypt1 subunit of MP by ROCK, downstream of Wnt3a. Treatment of differentiating mouse ESCs with Wnt3a resulted in exclusion of Nkx2.5 from the nucleus and a subsequent failure to undergo terminal differentiation into cardiomyocytes. This likely represents part of the molecular basis for Wnt-mediated inhibition of terminal differentiation of cardiomyocytes. Taken together, our results provide novel insight into the relationship between myogenic signalling cascades and downstream transcription factors and into how they function together to orchestrate the myogenic cell fate in stem cells.

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst stage embryo. They exhibit unlimited proliferation in culture and have the ability to differentiate into all three germ layers of the developing organism, a property defined as pluripotency. Previously it was reported that growth factor-bound protein 2 (Grb2) is required for differentiation of the epiblast, the embryonic tissue that harbours the pluripotent founder cells of the foetus. GRB2 is an adapter protein involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in response to extracellular signals. It has also been implicated in the activation of the phosphoinositol-3-kinase (PI3K) pathway in response to fibroblast growth factor (FGF) signaling. The work presented in this thesis examines the role of Grb2 in ES cells and describes previously unreported contributions of this adaptor protein in regulating ES cell growth and differentiation. It has been previously been shown by others that Grb2 deficient (Grb2-/-) cells grow relatively normally in ES growth medium containing serum. However, in serum free conditions (N2B27 medium) in this project, proliferation of Grb2-/- cells is reduced compared with wild type and “restored” Grb2-/- cells stably expressing a Grb2 cDNA mini gene. Under serum free conditions, Grb2-/- cells grow in tight, refractive colonies. Nanog expression was uniformly upregulated, in contrast to the heterogeneous pattern reported in serum-based medium. Colony expansion on the substratum appears to be compromised, although there is no apparent defect in the initial attachment of Grb2-/- cells. Cell cycle analysis indicates that the slower growth of Grb2-/- cells in serum free medium could be due to lengthening of the G1 phase of the ES cell cycle. In an attempt to identify the signalling deficiency responsible for the growth defect of Grb2-/- cells, MAPK activation was restored by two methods, PMA a ligand that bypasses the requirement for Grb2, and Raf-ER, a conditionally regulated component of the MAPK pathway that acts downstream of Grb2 in the MAPK pathway. Although both approaches increased MAPK signalling they were unable to rescue the growth defect. This suggests that MAPK is not required or alone is not sufficient. Inhibition of Glycogen synthase kinase 3 β (GSK3 β ) is known to augment growth of ES cells under MAPK inhibition. Surprisingly, GSK3 β inhibition did not enhance Grb2-/- cell growth. Under GSK3 β inhibition, Grb2-/- ES cells fail to thrive. It is hypothesised that under these conditions cells undergo hyper-self-renewal at the cost of growth. Grb2-/- ES cells are reported to exhibit limited differentiation potential. To examine the potency of Grb2-/- cells, these cells were subjected to embryoid body (EB) and monolayer differentiation. Analysis of EBs showed a loss of Gata4, Gata6 and endoderm marker gene expression. However, markers of ectoderm (Sox1, Pax6, MAP2), the late epiblast/nascent mesoderm (Brachyury) and markers associated with gastrulation (Twist and Snail) were expressed. Outgrowths of morphologically and immunohistochemically identifiable neuronal cells confirmed differentiation of ectodermal cell types, indicating Grb2 is not required for neuronal differentiation. However, beating cardiomyocytes could not be identified in Grb2-/- EBs, though readily found in restored Grb2-/- cells expressing the Grb2 cDNA. This suggests that there is an essential role for Grb2 in the mesoderm/cardiomyocyte differentiation pathway. This may be due to a defect in GATA factor expression since these factors are essential for cardiogenesis. In serum-free monolayer differentiation, Grb2-/- cells formed neuronal cells. Additional inhibition of the MAPK pathway using a small chemical inhibitor failed to prevent this differentiation. However, biochemical analysis of the cells indicates that this occurs when ERK activation is very low, indicating differentiation was not MAPK-independent. Grb2 mediates FGF-MAPK induced exit from the naïve ground state. These data suggest a Grb2-independent pathway can also facilitate this transition. Grb2 is dispensable for differentiation in to some lineages. However as differentiation of Grb2-/- ES cells is restricted, this indicates Grb2 is required for true pluripotency.

Cardiovascular disease is the leading cause of death worldwide. Due to the extremely low natural regeneration rate of heart muscle, development of new therapeutics directed towards heart repair is challenging. A potential approach to regenerate damaged heart is offered by cardiac tissue engineering. Specifically, it aims at engineering cardiac muscle in vitro and implanting it into the site of injury so that it can be integrated into the host tissue and restore the heart’s function. To ensure the effectiveness of this technique, the engineered tissue needs to recapitulate structural and functional properties of the native myocardium. Myocardium consists of laminar sheets of uniaxially aligned cardiac muscle cells (cardiomyocytes) wrapped around the heart. Therefore, achieving high cardiomyocyte alignment in engineered muscle is crucial. In this study we aimed at stimulating cardiomyocyte alignment by mimicking their niche in the embryonic heart. We hypothesized that recapitulating the extracellular cues that guide myocardial development in the embryo can guide cardiac tissue organization in vitro. To test this hypothesis, we imaged the structure of fibronectin – the most abundant protein in embryonic heart’s extracellular matrix (ECM) – and derived a 2D pattern from it that was then microcontact printed onto a substrate to guide cell alignment. We compared chick cardiomyocyte alignment on the biomimetic pattern and line patterns that have been extensively studied in the past. Results revealed a unique cell density-dependent response of cardiomyocytes to the biomimetic pattern that allowed us to elucidate the role of cell-cell and cell-ECM interactions in cardiomyocyte alignment on fibronectin patterns by looking at the effect of local pattern features on alignment and inhibiting N-cadherin-based cell-cell junctions. Further, to engineer more clinically relevant tissues, we differentiated human induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) into cardiomyocytes and seeded them onto the fibronectin patterns. Cardiac tissues produced with these cells showed significant differences compared to the chick tissues due to their immature phenotype. We showed that co-culture with cardiac fibroblasts (CFBs) as well as maturation of iPSC-derived cardiomyocytes (iPSC-CMs) increased tissue alignment, indicating the important role of both of these factors in developing novel methods to engineer functional cardiac tissues.

Class of 2009 Abstract / OBJECTIVES: The objective of this study was to characterize the Hyaluronidase (HYA) gene family throughout gestational development of murine hearts to provide greater insight regarding its role in cardiac morphogenesis. METHODS: Microdissection of murine embryos was accomplished to extract embryonic heart tissue. RNA was extracted using the standard Trizol protocol. cDNA templates were created using a standard protocol. Polymerase chain reaction (PCR) was used to verify presence of HYA, isolate a sample for insertion into a cloning plasmid to make a recombinant clone. A TOPO cloning reaction followed by a double DNA digest was accomplished to verify gene sequencing and orientation in the clone. SYBR Green real time RT- PCR was used to quantify gene expression relative to 18S RNA. RESULTS: RT-PCR provided qualitative data indicating HYA1, HYA2, and HYA3 are present at all observed time points (E8.5, E9.5, E10.5, E11.5, E12.5, E13.5, E14.5, E15.5, and E16.5). Real time RT-PCR data results characterizing relative expression for HYA2: E9.0 (Rel. Exp. = 1.00; SD = 0), E10.5 (Rel. Exp. = 1.33; SD = 0.577), E12.5 (Rel. Exp. = 2.00; SD = 0), E13.5 (Rel. Exp. = 2.66; SD = 0.577), E14.5 (Rel. Exp. = 3.00; SD = 0), E15.0 (Rel. Exp. = 2.00; SD = Error). CONCLUSIONS: HYA1, HYA2, and HYA3 are present at al time points observed in embryonic heart tissue. Relative expression of HYA2 progressively increased from E9.0 until E14.5 and then started tapering downward at time point E15.0.

The onset of agriculture about 12,000 years ago has had a major influence on the biodiversity of plants and animals. Unfortunately, the rapid changes in agricultural practices that has occurred in recent times has negatively affected many farmland species. One such species is the grass snake (Natrix natrix), which has been reported to decline in many parts of Europe, including Sweden. The grass snake is unique, not only in that it is the most northerly distributed oviparous reptile in the world, but also because of its habit of using anthropogenic heat sources such as manure heaps and composts as nesting-sites. Unfortunately changes in manure management and abandonment of farmlands have resulted in a decline and fragmentation of these environments. This may pose a threat for the northernmost populations of the grass snake, because natural nests in these areas may not provide sufficient heat for the eggs to hatch. The eggs and embryos of reptiles are highly sensitive to incubation temperatures, which can influence not only hatching success but also many phenotypic traits in the hatchlings. In this thesis I used a series of laboratory and field experiments to investigate the importance of anthropogenic heat sources for the reproductive ecology of cold-climate populations of grass snakes.  More specifically, I aimed to investigate thermal regimes of nests and how they influence embryonic development and offspring traits associated with survival and fitness. The results showed that manure heaps and composts are significantly warmer than potential natural nests and that natural nests do not provide sufficient heat to sustain embryonic development. Further, manure heaps were warmer and more constant in temperature than composts, resulting in higher hatching success and earlier hatching in manure heaps. The higher thermal variability in composts increased the frequency of abnormalities that are likely to negatively affect survival and fitness. In conclusion, this thesis shows that the use of anthropogenic heat sources has enabled grass snakes to expand their range farther north than any other oviparous reptile and that the thermal dichotomy in the primary nesting environments used by grass snakes contribute to important life-history variation in this species. These findings have important implications for conservation of reptile populations in general and grass snakes in particular.

The first transplantable hematopoietic stem cells (HSC) arise in the aorta-gonad mesonephros region (AGM) during early stages of embryo development. Specifically, ventral aspect of embryonic dorsal aorta (DA) contains HSC that upon transplantation into irradiated recipients can reconstitute all lineages of the haematopoietic system [Medvinsky et al. 1993; Muller and Medvinsky, 1994; Medvinsky and Dzierzak, 1996; Cumano et al., 1996; Tavian et al., 1996; Peault and Tavian, 2003; Taoudi and Medvinsky, 2007; Ivanovs et al., 2011, 2014]. The ventral aspect of DA bears so-called intra-aortic cell clusters (IAC), the appearance of which coincides with the emergence of HSC [Babovic and Eaves, 2014; Bhatia, 2007; Boisset et al., 2010, 2011; Bollerot et al., 2005; de Bruijin et al., 2002; Bertrand et al., 2010]. According to recent reports, HSC are a heterogeneous population of cells [Dykstra et al., 2007; Seita and Weissman, 2010; Muller-Sieburg et al., 2012]. It is unclear whether all HSC precursors originate from the same location, for example, DA lining, IAC or sub-aortic tissues; or HSC precursors migrate into DA lining from other parts of the embryo [Tavian et al., 1999; Yoder et al., 1997; Oberlin et al., 2002; Peault and Tavian, 2003; Dzierzak, 2003; Samokhvalov et al., 2007; Medvinsky et al., 2011]. To elucidate ontogeny of early HSC precursors (pro-HSC), two approaches were applied in this PhD project. First, we mapped potential pro-HSC in pre-circulation mouse embryos (embryonic day 6-8.5, E6-E8.5). We defined potential pro-HSC as cells co-expressing the transcription factor Runx1, endothelial markers (VE-Cad or CD31) and/or haematopoietic markers (CD45, CD41) [Oberlin et al., 2002; de Bruijn and Dzierzak, 2012; Liakhovitskaia et al., 2009, 2014]. In E6-E8 mouse embryo, prospective pro-HSC were found to be located in chorionic plate, yolk sac and in allantoic core domain. In early somitic mouse embryo (E8-8.5) cells with pro-HSC phenotype (Runx1+CD31+CD41+) were found to be in cell clusters in forming vessel of confluence and in nascent dorsal aortae lining. Pro-HSC are not directly transplantable [Cumano et al., 1996., 2001; Godin et al., 1993; 1995; Batta et al., 2016; Matsuoka et al., 2001; Nishikawa et al., 1998]. Therefore, cells and tissues containing prospective pro-HSC were initially matured using several in-vitro culture systems. According to our results, E8 mouse embryo pro-HSC are only preserved in explant cultures, but not in co-aggregate cultures with stroma cells. After culture, cells were transplanted into sub-lethally irradiated recipients. Six weeks after transplantation 19 out of 82 transplanted recipients had donor derived blood cells' chimerism at the level of 0.1-0.3%. Forty six percent of these grafts were derived from rostral part of the embryo tissues (head, heart, upper somites). Only one out of 82 recipients had donor cells contribution above 1% (1.2 %). This recipient was engrafted with cells derived from the E8 mouse embryo head and heart region. Recipients having blood chimerism at the range of 0.1-0.3% had mainly lymphoid donor derived cells in their peripheral blood. The only recipient showing the high donor cells contribution (1.2%) had contribution mainly to myeloid lineage. Recorded low levels of blood chimersims are in line with those reported by Rybtsov et al. (2014) for early E9 mouse embryos. Donor derived cells formed clearly distinguishable populations on cytometry plots. This population of cells were absent from control engraftment experiments with carrier cells only. Previously, lymphoid potential was detected in paraaortic spnanchnopleura (P-Sp) of E8.5-9 mouse embryos, but not in E8 mouse embryos (0-5 somites, pre-circulation) and later in yolk sac [Cumano et al., 1996; Nishikawa et al., 1998; Fraser et al., 2002; Yokota et al., 2006]. However, prior works used different criteria to establish recipient reconstitution. Therefore, it is possible that recipients repopulated with E8 derived cells at the level of 0.1% were not considered as repopulated and hence, presence of lymphoid lineage precursors was overlooked in early somitic mouse embryos. The only recipient showing substantial myeloid cells contribution (73% Mac1+Gr1+ cells of donor derived cells) received engrafted cells from an older (6-13 sp) embryo and therefore potentially has yolk sac derived myeloid cells. Yolk sac cell contribution to myeloid lineage, specifically to the brain microglia was reported in prior works [Samokhvalov et al., 2007]. Our data show that early E8 AGM cells do not expand in in vitro conditions. While in AGM, cells from E9 mouse embryo expand in culture [Rybtsov et al., 2014]. We have analysed Runx1 expression pattern and dorsal aorta morphology at the time when E9 HSC precursors acquire ability to expand in in vitro culture. Runx1 expression becomes clearly polarised at the time point (22-26 sp), when paired dorsal aortae fusion is initiated. We envision that intimate connection between DA fusion events and induction of pro-HSC maturation exists. According to prior reports, Bmp, Shh and VEGF signalling regulate DA fusion [Garriock et al., 2010]. Thereofore, to enhance in vitro HSC maturation system, DA fusion triggers (for example, Bmp4) might be added to culture. Since, pro-HSC maturation methods established to date are not efficient to expand and differentiate E8 pro-HSC into potent HSC, another approach had to be implemented to study HSC ontogeny. The second approach we utilized was to trace the origin of HSC in chicken embryo, starting from the very beginning of cell fate specification, i.e. from gastrulation stages. Chick embryo haematopoiesis is similar in both human and mouse: precursors of HSC arise in the embryo proper in AGM, and IAC are formed in DA ventral aspect [Dieterlen-Lièvre, 1975; Dieterlen-Lièvre and Martin, 1981; Dieterlen-Lièvre and Jaffredo, 2009; Jaffredo et al., 2000; Le Douarin and Dieterlen-Lièvre, 2013]. In contrast to mammals, chick embryo develops ex vivo, making direct labelling and cell tracing possible. We aimed to identify cells giving rise to regions of DA that produce IAC. Therefore, segments of primitive streak (PS) were labelled with lipophilic dyes or by substituting segments of host PS with PS sections derived from transgenic (GFP+) stage matched chicken embryos. Our results show that in an 18-25h chicken embryo (Hamburger and Hamilton developmental stage 4-6, HH4-6) cells giving rise to DA ingress through the wide region of PS (35-60% of its length) [Hamburger and Hamilton, 1951]. We identified that the section of DA producing HSC is formed by cells ingressing through PS in region of 40-55% of its length at 18-25h of chick embryo development. Regardless of the embryo development stage (HH4-6), in chimeras grafted at 40-55% of PS length, GFP+ cells contributed to DA and to the IAC. Within GFP+ labelled areas, we observed clusters consisting entirely of GFP+ and clusters having a mixture of GFP+ and GFP- cells. Entirely GFP+ clusters were found in the stretch of DA that had the entire aortic endothelial lining labelled. Clusters formed on the mosaic (GFP+/GFP-) aortic endothelium also had mosaic nature. According to our data, multiple descendants of PS contribute to the same stretch of dorsal aorta. This explains mosaicity of dorsal aorta lining and IAC labelling. Since we encountered clusters with mixture of GFP+ and GFP- cells, we conclude that IAC are not clonal formations. Mosaicity of IAC also does not exclude a scenario when cells migrate in and out of a cluster. Further tracing experiments are required to establish HSC nature of cells within a cluster.

This thesis presents the development of microfluidic devices designed to facilitate research into mouse embryonic stem cells (ESCs). ESCs are a well-studied cell, largely due to their pluripotent nature, meaning they are able to differentiate into all cell types of the body and may self-renew indefinitely in appropriate culture conditions. ESCs, along with many other lines of biological enquiry, are increasingly studied with the use of micro uidic technology which enables fine tuning of physical and chemical environments unachievable on the macro scale. Two varieties of microfluidic technology are presented in this thesis, one for high- resolution mechanical phenotyping of ESCs and the second as a novel in-chip culturing platform to study cellular transitions. Chapter 1 presents a broad introduction to ESCs and biological enquiry with microfluidics, aimed to underpin the following Chapters. Chapters 2 and 3 present self-contained projects, thus each include a motivation and introduction section more specific than that presented in Chapter 1. These Chapters also contain their own methods, results and conclusion sections. Finally, Chapter 4 presents a summary of the work performed along with an outlook of upcoming investigations. In Chapter 2, I present a microfluidic device developed and utilised in collaboration with Christophe Verstreken (Department of Physics, University of Cambridge), which has been used to apply a mechanical stress to live cells enabling measurement of their nuclear deformability. The device facilitates detection of both nucleus and cytoplasm which can then be analysed with a custom-written MATLAB code. Quantitative measurements of nuclear sizes and strains of ESCs indicated a negative Poisson ratio for nuclei of cells cultured in specific medium conditions. Furthermore, we demonstrate that the device can be used to physically phenotype at high-throughput by detecting changes in the nuclear response after treatment with actin depolymerising and chromatin decondensing agents. Finally, we show the device can be used for biologically relevant high-resolution confocal imaging of cells under compression. The work from this chapter is presented in Hodgson et al. [1]. In Chapter 3, I present a novel microfluidic platform developed in collaboration with Prof. Austin Smith and Dr Carla Mulas (Centre for Stem Cell Research, Cambridge). The developed platform enables individual ESCs to be cultured under continued observation as they exit their pluripotent stem cell state. Each cell within the device may be extracted from the chip at any time for further investigation without disturbing other cells. Assessing the transition from the stem cell state in individual cells is paramount if we are to understand the mechanisms of pluripotency.

The decisions stem cells make impact both the development of adult vertebrates and systems within the body that require cellular replenishment to sustain life. Regardless whether a stem cell remains quiescent, divides, differentiates, or undergoes apoptosis—these processes are precisely controlled by internal gene regulatory networks that are instructed by external stimuli. The exact mechanisms governing stem cell fate are not completely understood. These studies explore new ways in which cell fate is mediated. Through a study of mitochondrial content in human embryonic stem cells (hESCs) and their differentiated progeny, we discovered differences in mitochondrial morphologies. Mitochondria began as elongated and networked structures in self-renewing conditions and changed their shape after differentiation. The addition of external growth factors that direct hESCs toward the definitive endoderm (DE) lineage promoted mitochondrial fragmentation, which was mediated by the mitochondrial fission machinery. Globular, punctate mitochondria were observed prior to the induction of the DE-specific transcriptional program. Differentiation of hESCs to other lineages did not result in any mitochondrial shape changes. Thus, mitochondrial fission in differentiating hESCs, an internal cellular process, is induced by DE-inducing external stimuli, an effect that was lineage specific. In a second study, we investigated the role of the splenic environment in the development of the blood system—during hematopoiesis. The spleen made a distinct contribution to hematopoiesis, a process predominantly attributed to the bone marrow. We discovered a previously unidentified population of cells, uniquely represented in the mouse spleen that could develop into erythrocytes, monocytes, granulocytes, and platelets. These multipotent progenitors of the spleen (MPPS) expressed higher levels of the transcription factor, NR4A1 compared to their bone marrow counterparts and relied on NR4A1 expression to direct their cell fate. The activation of NR4A1 in MPPS biased their production of monocytes and granulocytes in vitro whereas NR4A1-deficient MPPS over-produced erythroid lineage cells in vivo. Together, these data suggest the splenic niche supports distinct myeloid differentiation programs of multi-lineage progenitors cells. Both studies identify new mechanisms by which external stimuli regulate internal mechanisms of cell fate. These insights provide a better understanding of stem and progenitor cell differentiation that have the potential to impact cellular replacement therapies.

The aim of this project is to investigate genetic mechanisms of early development of vertebrate embryos using model fish species. Zebrafish (Danio rerio) and medaka (Orizias latipes) have been used extensively for molecular genetics and developmental biology studies because these fish produce many eggs, which can be manipulated from the 1 cell stage and are ideally suited for analysing gene expression, function, and embryonic phenotypes. These species have already been extensively used to generate many mutants which show clear phenotypes during early embryonic development. The development of other model species for mutant screening and analyses is likely to provide scope to analyse gene function from uncharacterised/under-characterised genes. Therefore we have developed and tested a small number of early developmental mutants from the mangrove killifish (Kryptolebias marmoratus). To achieve my aim, embryos from zebrafish, medaka and the mangrove killifish have been used as models to study gene function and understand the molecular mechanisms for early patterning genes. We focused in particular on development of neural ectoderm and non-neural ectoderm (epidermis) and anterior-posterior patterning (head, trunk and tail development). As different model animals have different advantages, we used these model animals for different purposes. Zebrafish and medaka were used with chemical treatment (specific inhibitors of target genes) and morpholino analyses because they give many synchronized eggs every morning allowing highly replicated analyses. On the other hand, the mangrove killifish were used for developing and testing novel mutants and associated loss (or gain) of gene function. Firstly, zebrafish was used to study maternal fibroblast growth factor (FGF) signaling at pre-maternal zygotic transition (Pre-MZT) and consequent neural induction at the gastrula stage (Chapter 3). This study found the important role of acquiring maternal FGF signaling in stem cells to achieve neural induction during the zygotic gene expression stage. An FGF signaling inhibitor SU5402 was tested using RNA–seq, ATAC-seq, in.situ hybridization and immunohistochemistry methods. Through these techniques, we found that the maternal FGF signaling provides competence to the ectodermal stem cells for neural induction possibly via epigenetic modification of histone trimethylation. To examine the role of a specific FGF molecule (FGF2), gene knockdown was conducted to study fgf2 gene function during early development in zebrafish (Chapter 4). In situ hybridization and immunostaining with tissue-specific markers at the gastrula stage were used to discover a novel role for fgf2 in development of the epidermis. The final stage of my project involved characterization of mutations underlying two mutant phenotypes (short tail/stl and ball tail/stl), that exhibit defects in tail development using the self-fertilizing mangrove killifish (Chapter 5). Using a small scale RNA-seq, the mutated genes responsible for the stl and btl mutations were instantly identified as noto and msgn1 respectively. The mutant phenotype was phenocopied by morpholino injections in medaka. This study revealed crucial roles of the two genes in tail bud development. Defects of these genes affected the motility of progenitor cells in the tail bud by suppressing cell translocation to the axial mesoderm in the noto mutation and to the paraxial mesoderm in the msgn1 mutation. The study demonstrated similarity of gene function and redundancy in the mangrove killifish and medaka that is different from the function of these genes in zebrafish, revealing the importance of research on different model animals to fully characterise the gene function. From these data, it can be considered that mangrove killifish is very powerful model for mutation screening, suggesting that this animal model can be applied in various genetic studies alongside or in addition to other vertebrate models.

Stem cell pluripotency and self-renewal are two important attributes of human embryonic stem cells which have led to enhanced interest in stem cell research. Understanding the mechanisms that underlie the regulation and maintenance of these properties is imperative to the clinical application of stem cells. Pluripotency and self-renewal are regulated by different genes, transcription factors and other co-factors such as FoxD3 and Klf4. Oct4, Nanog and Sox2 are central to the stem cell regulatory circuitry. They form interactions with co-factors to promote cell proliferation and inhibit differentiation by negatively regulating differentiation markers. However, there are other novel pluripotency associated factors yet to be studied. In this study, bioinformatics and functional analyses were employed to identify a potential pluripotency gene called YY1AP1 from our lab's pre-existing microarray data. YY1AP1, a transcription regulatory gene, showed consistent down-regulation with induced cell differentiation. It was further investigated. First, its co-localization with Oct4 in both hESCs and iPSCs was confirmed by immunofluorescence staining. Knockdown experiments were then performed on this gene to investigate effects of knocking it down on gene expression in hESCs. Knocked-down cells were characterized for markers of pluripotency and differentiation at the transcript level. Results showed a down-regulation of pluripotency genes with no specific promotion of any of the germ layer markers. Gene expression at the protein level in knocked down cells was then assessed for YY1AP1, and its binding partner YY1, and pluripotency markers. Results showed that proteins of YY1AP1, YY1, Oct4, Nanog and CTCF were down regulated while the tumour suppressor gene protein, p53, was up-regulated in YY1AP1 deficient stem cells. Protein to protein interaction studies showed that YY1AP1, YY1, Nanog and CTCF proteins directly interacted with each other. Differentiation of YY1AP1deficient cells into EBs led to an almost complete shutdown of all gene expression, an indication that the cells did not form 'real' EBs. Differentiation of YY1AP1 ablated cells did not support any lineage promotion either. These results suggest a potentially new role for YY1AP1 in proliferation and self-renewal of stem cells through its possible direct binding to CTCF or its indirect binding to CTCF in complex with YY1.